1. Field of the Invention
PEPTIDIC PRODUCT, PROCESS and COMPOSITION The present invention relates to a peptidic product comprising an active tetrapeptide Acetyl-glutamyl-glutamyl-glutamyl-aspartyl (SEQ ID No. 5) associated with a vector which allows the introduction of said tetrapeptide into a cell.
The present invention is also related to pharmaceutical compositions comprising said peptidic product and to its use for the prevention and/or the treatment of wound contractions, hypertrophic scars, fibromatosis, in particular the so-called Dupuytren disease and fibrotic conditions (in particular lung fibrosis).
2. Description of the Related Art
Skalli et al. (J. of Cell Biology Vol. 103 No. 6, pp. 2787-2796 (1986)) describe that an antibody (anti-alpha SM-1) recognising exclusively the alpha smooth muscle (xcex1-SM) actin could be selected and characterised after immunisation of BALB/c mice with the amino terminal synthetic decapeptide of xcex1-SM actin coupled to a carrier molecule.
It is also known that the xcex1-SM actin is transiently expressed in myofibroblast during experimental wound healing (Darby et al., Laboratory Investigation Vol. 63, 21-29 (1990)). Chaponnier et al. (J. of Cell Biology Vol. 130 No. 4, 887-895 (1995)) describes the blocking effects of an amino terminal decapeptide of a-SM actin (AcEEEDSTALVC) (SEQ ID No: 6) on the binding of specific monoclonal antibody anti-alpha SM-1. This document indicates that the portion AcEEED (SEQ ID No: 5) is the epitope for the anti-alpha SM-1.
The anti-alpha SM-1 is the first monoclonal antibody described to be specific for a single actin isoform and is a reliable tool for the study of vascular SM cells and myofibroblast phenotypic modulation in physiological and pathological processes.
This document shows that said antibody (especially its Fab fragment) increases in vitro xcex1-SM actin polymerisation. Said document also suggests that the tetrapeptide AcEEED (SEQ ID No: 5) may be used for the study of the functions of the xcex1-SM actin in cells such as SM cells, fibroblasts, myofibroblasts or myoepithelial cells in physiological and pathological situation during which xcex1-SM actin expression is modulated. Said document also suggests that the tetrapeptide AcEEED (SEQ ID No: 6) inhibits the incorporation of xcex1-SM actin into stress fibers in the culture of SM cells when using microinjection technology.
It is also shown that an active turnover of xcex1-SM actin into stress fibers exists and that AcEEED (SEQ ID No: 5) traps a protein interacting with the amino-terminal sequence of xcex1-SM actin and activating polymerization (Chaponnier, C. et al., Experientia, 1995, 51, A62).
Jxc3xa4rlebark, L. et al. (Biochemical and Biophysical Research Communications, 1996, 229 (2), 363-369), disclose that some peptidyl derivatives of adenosine 5xe2x80x2-carboxylic acid have inhibitory effects in certain P2 purinoceptor-carrying biological systems (gliome and smooth muscle cell lines and isolated smooth muscle tissue preparations).
The present invention aims to provide a peptidic product comprising the specific tetrapeptide AcEEED (SEQ ID No: 5) which allows its introduction into a cell, in order to interfere, in vitro and in vivo, with xcex1-SM-actin organisation in stress fibers.
Another aim of the present invention is to provide a pharmaceutical composition comprising said peptidic product in order to treat and/or to prevent various diseases related to xcex1-SM actin expression such as wound contraction, hypertrophic scars, fibromatosis, in particular Dupuytren diseases, and fibrotic condition (in particular lung fibrosis).
The present invention is related to a peptidic product comprising the active tetrapeptide Acetyl-glutamyl-glutamyl-glutamyl-aspartyl (SEQ ID No: 5) (identified hereafter by the formulation AcEEED) associated with a chemical entity which is able to introduce said tetrapeptide into a cell.
According to the invention, in the active tetrapeptide Ac means Acetyl, E means glutamic acid residue, D means aspartic acid residue.
According to the invention, by a chemical entity we understand a vector.
According to the invention, by a cell we understand in vivo cells, such as cells composing the organs or tissues of living animals or humans, or microorganisms found in living animals or humans; or in vitro cells,such as cultured animal cells, human cells or microorganisms. Preferably it means human cells, and more preferably fibroblastic or smooth muscle cells.
According to a first embodiment of the present invention, the chemical entity can be a liposome, a lipidic vesicle made of lipids and/or fatty acids or a cationic vesicle such as the one described by Gao X. and Huang L. (Cationic Liposomes - Mediated Gene Transfer, Gene Therapy, pp. 710-722 (1995)) and/or a mixture thereof.
According to a second embodiment of the present invention, the chemical entity is a polypeptide having preferably at least about 8 amino acids, preferably between 8 and 40, more preferably between 10 and 30 amino acids.
According to another embodiment of the present invention, said polypeptide is the tat mediated protein, having advantageously a capacity of binding with membranous receptor(s) of specific cell(s).
The preferred linking of a chemical entity like the tat mediated protein to the tetrapeptide according to the invention for the delivery of heterologous peptides into a cell, is described in the International Patent Publication WO 94/04686 incorporated herein by reference. The best results have been obtained with the tat mediated protein of formula tat37-72, tat37-58, tat38-58GGC, tatCGG47-58, tat47-58GGC and tatxcex94C.
According to a preferred embodiment of the present invention, the chemical entity is a homeobox peptide, preferably the pAntp peptide described in the International Patent Publication WO 97/12912, also incorporated herein by reference. The best results have been obtained with the peptide pAntp[43-58], and the peptide pAntp[43-58]-Pro50. The peptide pAntp[43-58] means Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys, (SEQ ID No: 7) wherein, for this description, the first amino acid Arg is named amino acid in position 43 and the last amino acid Lys is named amino acid in position 58. The peptide pAntp[43-58]-Pro50 (SEQ ID No: 7) represents the same sequence of pAntp[43-58]wherein Gln has been substituted by Pro in position 50.
The present invention relates also to the preparation method of such product, using known methods by the person skilled in the art such as preparation methods of lipidic vesicles (Martin F. G., Pharmaceutical Manufacturing of Liposomes, Praveen Tyle Ed., Specialised Drug Delivery Systems, Marcel Decker, N.Y. (1993)), automatic peptides synthesis, cross-linking between olecules.
Possibly, the tetrapeptide is linked through a spacer, preferably a peptidic spacer, to the chemical entity. Said spacer is preferably an amino acid or a peptide comprising no more than 5 amino acids. Preferably, said amino acid used as a spacer is a Lysyl residue (xe2x80x9cKxe2x80x9d) to which the AcEEED (SEQ ID No: 7) tetrapeptide is linked through the xcex1-amino group, or through the xcex5-amino group, or a Cystinyl residue (meaning two Cysteinyl residues (xe2x80x9cCxe2x80x9d) linked by their xcex2 sulfide functions forming a disulfide link represented by xe2x80x9cC-[S-S]-Cxe2x80x9d) to which the AcEEED (SEQ ID No: 5) tetrapeptide is linked through the xcex1-amino group, or xcex2-Alanyl-xcex2-Alanyl dipeptide (xe2x80x9cxcex2-A-xcex2-Axe2x80x9d) to which the AcEEED (SEQ ID No: 5) tetrapeptide is linked through the xcex5-amino group, or through a Lysyl residue (xe2x80x9cKxe2x80x9d) to which the AcEEED (SEQ ID No: 5) tetrapeptide is linked through the xcex5-amino group and a BiotynylGG residue (meaning a biotinyl functionnal group linked through its carboxylic function to two successive glycyl residues) to which the AcEEED (SEQ ID No: 5) tetrapeptide is linked through the xcex1-amino group.
The best results have been obtained with the AcEEED (SEQ ID No: 5) tetrapeptide linked through the xcex1-amino group, or through the xcex5-amino group of a Lysyl residue.
According to the invention, the peptidic product is preferably selected from the group consisting of
xcex5-(AcEEED)-K-(pAntp[43-58]), (SEQ ID No: 1)
AcEEED-xcex2-A-xcex2-A-(pAntp[43-58]), (SEQ ID No: 2)
AcEEED-K-(pAntp[43-58]), (SEQ ID No: 2)
AcEEED-xcex2-A-xcex2-A-(pAntp[43-58]Pro50), (SEQ ID No: 2)
AcEEED-K-(pAntp[43-58]-Pro50), (SEQ ID No: 1)
AcEEED- (pAntp[43-58]-Pro50), (SEQ ID No: 3)
AcEEED-(pAntp[43-58]), (SEQ ID No: 3)
AcEEED-C-[S-S]-C-(pAntp[43-58]), (SEQ ID No: 2)
AcEEED-C- [S-S]-C- (pAntp[43-58]-Pro50) (SEQ ID No: 2)
xcex1-(BiotynylGG),xcex5-(AcEEED)-K-(pAntp[43-58]), (SEQ ID No: 4)
xcex5-(AcEEED)-K-(pAntp[43-58]-Pro50), (SEQ ID No: 1)
xcex1-(BiotynylGG),xcex5-(AcEEED)-K-(pAntp[43-58]-Pro50), (SEQ ID No: 4)
The peptidic product is more preferably selected from the group consisting of
AcEEED-K-(pAntp[43-58]), represented by the following structure: xcex5-(AcEEED)-K-RQIKIWFQNRRMKWKK-OH (SEQ ID No: 1) (identified hereafter by the term xe2x80x9cproduct 1xe2x80x9d);
AcEEED-K-(pAntp[43-58]), represented by the following structure: AcEEED-K-RQIKIWFQNRRMKWKK-OH (SEQ ID No: 1) (identified hereafter by the term xe2x80x9cproduct 2xe2x80x9d);
AcEEED-xcex2-A-xcex2-A-(pAntp[43-58]), represented by the following structure: AcEEED-xcex2-A-xcex2-A-RQIKIWFQNRRMKWKK-OH (SEQ ID No. 2) (identified hereafter by the term xe2x80x9cproduct 3xe2x80x9d);
AcEEED-(pAntp[43-58]), represented by the following structure: AcEEED-RQIKIWFQNRRMKWKK-OH (SEQ ID No: 3) (identified hereafter by the term xe2x80x9cproduct 4xe2x80x9d);
AcEEED-C-[S-S]-C-(pAntp[43-58]), represented by the following structure: AcEEED-C-[S-S]-C-RQIKIWFQNRRMKWKK- OH (SEQ ID No: 2) (identified hereafter by the term xe2x80x9cproduct 5xe2x80x9d);
xcex1-(BiotynylGG),xcex5-(AcEEED)-K-(pAntp[43-58]), represented by the following structure: xcex1-(Biotinyl-GG), xcex5-(AcEEED)-K-RQIKIWFQNRRMKWKK-OH (SEQ ID No: 4) (identified hereafter by the term xe2x80x9cproduct 6xe2x80x9d);
xcex5-(AcEEED)-K-(pAntp[43-58]-Pro50), represented by the following structure: xcex5-(AcEEED)-K-RQIKIWFPNRRMKWKK-OH (SEQ ID No: 1) (identified hereafter by the term xe2x80x9cproduct 7xe2x80x9d); xcex1-(BiotynylGG),xcex5-(AcEEED)-K-(pAntp[43-58]-Pro50), represented by the following structure: xcex1-(BiotinylGG), xcex5-(AcEEED)-K-RQIKIWFPNRRMKWKK-OH (SEQ ID No: 4) (identified hereafter by the term xe2x80x9cproduct 8xe2x80x9d).
The best results have been obtained with the product xcex1-(BiotynylGG),xcex5-(AcEEED)-K-(pAntp[43-58]-Pro 50) (SEQ ID No: 4) and the product xcex5-(AcEEED)-K-(pAntp[43-58]).
In products 1 to 8, xcex1-( )-K- means that functionnal group in brackets is linked on the alpha-amino group of lysyl residue; xcex5-( )-K- means that functionnal group in brackets is linked on the epsilon-amino group of lysyl residue; -xcex2-A represents beta-alanyl residue; and -C-[S-S]-C represents two cysteinyl residues linked through their xcex2 sulfide function, forming a disulfide bridge. Products 1, 5 to 8 are not linear, are branched sequences; products 2, 3 and 4 are linear, are unbranched sequences. In the sequence listing, product 2 is represented by the sequence SEQ ID NO:1; product 3 is represented by the sequence SEQ ID NO:2, where Xaa means xcex2 Ala; and product 4 is represented by the sequence SEQ ID NO:3.
Product 1 is xcex5-(Acetyl Glu Glu Glu Asp) Lys Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys OH (SEQ ID No: 1).
Product 2 is Acetyl Glu Glu Glu Asp Lys Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys OH (SEQ ID No: 1).
Product 3 is Acetyl Glu Glu Glu Asp xcex2Ala xcex2Ala Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys OH (SEQ ID No: 2).
Product 4 is Acetyl Glu Glu Glu Asp Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys OH (SEQ ID No: 3).
Product 5 is Acetyl Glu Glu Glu Asp Cys[S-S]Cys Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys OH (SEQ ID No: 2).
Product 6 is xcex1-(Biotinyl Gly Gly) xcex5-(Acetyl Glu Glu Glu Asp) Lys Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys OH (SEQ ID No: 4).
Product 7 is xcex5-(Acetyl Glu Glu Glu Asp) Lys Arg Gln Ile Lys Ile Trp Phe Pro Asn Arg Arg Met Lys Trp Lys Lys OH (SEQ ID No: 1).
Product 8 is xcex1-(Biotinyl Gly Gly) xcex5-(Acetyl Glu Glu Glu Asp) Lys Arg Gln Ile Lys Ile Trp Phe Pro Asn Arg Arg Met Lys Trp Lys Lys OH (SEQ ID No: 4).
The present invention concerns also a process for preparing the peptidic product comprising the active tetrapeptide AcEEED (SEQ ID No: 5) associated with a chemical entity.
The peptidic product of the invention can be prepared using known methods by the person skilled in the art, such as the step-wise solid-phase synthesis, the straightforward synthesis of a peptide, the synthetic or biologic preparation of peptide (described for examples in xe2x80x9cThe peptides, Analysis, Synthesis, Biologyxe2x80x9d; vol. 1 to 9, S. Udenfriend, J. Meienhofer, eds, 1977 to 1987, Academic Press, New-York; or in xe2x80x9cMethods in Molecular Biologyxe2x80x9d, vol. 35, Peptide Synthesis Protocols, M. W. Pennington and B. M. Dunn (Eds), 1994, Humana Press, Totowa N.J.).
The present invention concerns also a peptidic product comprising the active tetrapeptide AcEEED (SEQ ID No: 5) associated with a chemical entity for use as a medicament.
Another aspect of the present invention concerns a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier, vehicle or excipient and the peptidic product.
A medical preparation with the said pharmaceutical composition has been applied topically at an effective dose of 1-3 mg peptide/1 ml saline, resulting in the best efficacy according to the present invention.
For implementing the method of treatment of the invention, the pharmaceutical composition herein above described should contain an effective amount of the peptidic product. An effective amount can be readily determined by the use of conventional techniques and by observing results obtained under analogous circumstance. In determining the effective amount, a number of factors are considered including, but not limited to: the species of patient; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; and the use of concomitant medication.
An effective amount of the peptidic product in the composition of the invention will generally vary from about 1 microgram per kilogram of body weight per day (xcexcg/kg/day) to about 500 milligram /kg body weight/day. A posology (dose) of about 100 xcexcg to about 10 mg once or twice per day is preferred.
The medical preparation with the said pharmaceutical composition can be formulated in any form and administered by various routes or modes, including but not limited to intravenous injection, intramuscular injection, inhalation, transmucosal or transdermal delivery, nasal administration, oral administration, intestinal and rectal administration, according to clinical indications. A person skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the particular characteristics of the disease state to be treated, the stage of the disease, and other relevant circumstances.
The compositions of the invention can comprise the peptidic product in combination with at least one pharmaceutically acceptable carrier or excipient, the proportion and nature of which are determined by the solubility and chemical properties of the composition selected, the chosen route of administration, and standard pharmaceutical practice.
More particularly, the present invention contemplates pharmaceutical compositions consisting essentially of a therapeutically effective amount of the abovxcex5-described peptidic product in admixture or otherwise in association with one or more pharmaceutically acceptable carriers or excipients (collectively referred to hereafter as xe2x80x9ccarrierxe2x80x9d materials).
The carrier material may be a solid, semi-solid, or liquid material which can serve as a vehicle or medium for the active ingredient. Suitable carrier materials are well known in the art. The pharmaceutical compositions of the invention may be adapted for topical, injection or other use and may be administered to the patient in the form of injection, cream, ointment, powders, elixirs, syrups, solutions, suspensions, or the like. The pharmaceutical composition of the invention may also be adapted for rectal use and may then be administered to the patient in the form of suppositories.
The carrier material should be suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practice. Optionally, the pharmaceutical composition of the invention also contain a binder such as microcrystalline cellulose, gum or gelatine, a disintegrating agent such as alginic acid, a lubricant such as magnesium stearate, a glidant such as colloidal silicon dioxide, a sweetening agent such as sucrose or saccharin, a coloring agent or a flavouring agent such as peppermint or methyl salicylate.
Because of their easy administration, cream and solution represent the most advantageous topical dosage unit form.
In addition to the common dosage forms set out above, the compositions of the present invention may also be administered by controlled release means and delivery devices.
Such solutions or cream may also include one or more of the following adjuvants: a sterile diluent such as water for injection, physiologic saline solution, oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting of tonicity such as sodium chloride or dextrose. The preparation can be enclosed in ampules, or multiple dose vials made of glass or plastic.
The pharmaceutically acceptable excipient may vary according to the mode of administration and could be combined with an adjuvant in order to improve the therapeutical properties of the pharmaceutical composition or reduce its possible sidxcex5-effects.
Suitable pharmaceutically acceptable excipients are well known in the art and are selected according to the methods generally applied by pharmacists or doctors, and may include solid, liquid or gaseous, non-toxic pharmaceutically acceptable vehicles. The percentage of active product versus pharmaceutically acceptable excipient may vary within very large ranges, only limited by the tolerance and the possible sidxcex5-effects upon the animal, including the human, and by the frequency of administration.
The present invention concerns also the use of said pharmaceutical composition or said peptidic product for the manufacture of a medicament. The present invention concerns also the use of said pharmaceutical composition or said peptidic product for the manufacture of a medicament in the prevention and/or the treatment of a disease related to xcex1-SM actin expression into a cell (surexpression into a cell or expression in a cell which normally does not express xcex1-SM actin). Preferably, said disease is selected from the group consisting of wound contraction, hypertrophic scars, fibromatosis (especially Dupuytren""s disease) and fibrotic conditions (especially lung fibrosis).
The invention concerns also a method for manufacturing a medicament, wherein the peptidic product is used.
Another aspect of the present invention is related to a method of treatment and/or prevention of said diseases wherein said peptidic product or said pharmaceutical composition is administered to a mammal, in particular to a human, suffering from said diseases or presenting the symptoms of said diseases.